Received: 23 November 2021 | Revised: 20 September 2022 | Accepted: 22 September 2022 DOI: 10.1111/vcp.13200 B R I E F C O M M U N I C AT I O N Sample stability and heparin interference in ionized calcium and ionized magnesium measurements in horses using the Stat Profile Prime Plus co-­oximetry electrolyte analyzer Júlia Sanmartí1 | José Angel Robles-­Guirado2 | Eduard Jose-­Cunilleras1 | Anna Bassols2 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Abstract Universitat Autònoma de Barcelona, Background: The determination of iCa and iMg is important in veterinary medicine, Barcelona, Spain 2 Departament de Bioquímica i Biologia but their immediate determination in whole blood is not always possible. Their stabil- Molecular, Facultat de Veterinària, ity in other sample types and the existence of interferences must be evaluated before Universitat Autònoma de Barcelona, Barcelona, Spain its use. Objectives: We aimed to analyze the effects of storage time on the stability of iCa, Correspondence Anna Bassols, Departament de Bioquímica iMg, and other analytes in whole blood, plasma, and serum samples in horses and as- i Biologia Molecular, Facultat de sess the interference of heparin in these measurements. Veterinària, Universitat Autònoma de Barcelona, 08913 Bellaterra, Barcelona, Methods: Whole blood, heparin-­plasma, and serum samples from 10 horses were Spain. stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, and 168 hours after sample col- Email: anna.bassols@uab.cat lection using the Stat Profile Prime Plus Vet equipment (Nova Biomedical, Waltham, MA, USA). Results were analyzed by ANOVA or mixed-­effect models. Results: The concentration of iCa, iMg, total calcium (tCa), total magnesium (tMg), and the ratios iCa/tCa and iMg/tMg did not differ up to 168 hours when compared to the initial time. Total Ca, iMg, and tMg were not significantly different among sample types, but iCa concentrations were slightly but significantly lower in plasma. Freezing at −20°C did not affect iCa, iMg, tCa, and tMg. The pH increased in serum and plasma after 8 hours, and a mild negative correlation existed between plasma iCa concentra- tion and pH. A negative correlation was observed also between the ratios iCa/tCa or iMg/tMg and pH in plasma and serum. A significant decrease in iCa and iMg was detected when comparing homemade syringes at high heparin concentration (~200–­ 300 U heparin/mL) and commercial lithium-­heparin tubes (20–­30 U/mL). Conclusions: Samples stored at 4°C can be used to determine iCa and iMg concentra- tions up to 7 days after collection. Other metabolites are stable for up to 8 hours; hep- arin interference should be taken into account if using homemade heparin syringes. KEYWORDS equine, ionized calcium, ionized magnesium, point-­of-­care, sample stability, total calcium, total magnesium Júlia Sanmartí and José Angel Robles-­G uirado contributed equally to this work. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of American Society for Veterinary Clinical Pathology. Vet Clin Pathol. 2023;00:1–9.  wileyonlinelibrary.com/journal/vcp | 1 | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 SANMARTÍ et al. 1 | I NTRO D U C TI O N measure the activities of specific ions. Although several companies have developed point-­of-­care equipment that allows the measure- Calcium (Ca) and magnesium (Mg) are the main divalent cations in ment of iCa and other analytes in clinical settings, there is only one body fluids, and their measurement is important in understand- that allows the determination of iMg in whole blood, plasma, and ing certain body homeostasis imbalances. Both exist in plasma in serum that is marketed for veterinary use (Stat Profile Prime Plus three different forms: protein-­b ound, complexed with anions (eg, Vet, Nova Biomedical, Waltham, MA, USA). The measurement of carbonate, lactate, and phosphates), and free (ionized) divalent ions (calcium, magnesium, sodium, potassium, chloride) using this cations (iCa and iMg). In horses, the proportion of these calcium equipment is based on ISE. fractions is 40%–­45%, 5%–­10%, and 50%–­58% of total calcium, Considering this, the objectives of our study were to evaluate 1 respectively, while the magnesium fractions are 30%, 10%, and the effect of storage time on the measurement of iCa, iMg, and 60% of total magnesium. 2 The free forms of calcium and magne- other analytes after sample collection, and the potential heparin sium are the physiologically active components, responsible for interference in heparinized syringe samples from equine sam- triggering signaling transduction pathways, 3 and thus their con- ples, analyzed by the Stat Profile Prime Plus Vet analyzer (Nova centrations are more clinically relevant than their total concentra- Biomedical). tions, which are affected by total plasma protein concentration. Higher iCa and iMg concentrations are found in acidosis with lower levels in alkalosis, whereas total calcium and magnesium 2 | M ATE R I A L S A N D M E TH O DS 2,4 concentrations remain unchanged. Electrolyte imbalances are common in critically ill humans In the first experiment, blood was obtained from 10 horses using and animals, and hypocalcemia constitutes a frequent finding in a previously placed indwelling intravenous catheter. Horses were sepsis or endotoxemia. 5,6 In horses, electrolyte disturbances play client-­owned animals in the veterinary teaching referral hospital for an important role in diseases such as colic, renal failure, or sep- medical reasons, and the study did not require ethical permission sis. Specifically, severe enterocolitis is frequently accompanied since it was categorized as a nonexperimental clinical veterinary by hypocalcemia, which can manifest deleterious consequences practice by the institutional Ethics Committee on Animal and Human and may result in death. 6 Possible causes of hypocalcemia in ill Experimentation (CEEAH, UAB). The horses were hospitalized with horses include renal loss of calcium, sequestration of calcium in various medical conditions: five colic, one chronic weight loss, one the gastrointestinal tract due to inflammation, and impairment in distal limb laceration, one septic arthritis, one recurrent uveitis, calcium mobilization.1 Likewise, hypomagnesemia is possibly re- and one breeding mare accompanying a septic foal. There were lated to poor magnesium absorption in the inflamed bowel or ex- five geldings and five mares, and five cross-­bred, two Andalusians, 7 cessive urinary losses. Serum iMg concentration is also decreased and one Arabian, Friesian, and pony each. The horses ranged in age in horses with enterocolitis and hypocalcemia. 6 The role of iMg in 9.3 ± 5.2 years (average ± standard deviation [SD]). critically ill patients has received less attention than that of iCa, The sample size was calculated assuming a significance level but both electrolytes are closely associated. The effects of iMg on (alpha or P-­value) set at <0.05, the statistical power at >80%, a stan- mineral metabolism and its interaction with calcium homeostasis, dard deviation of 0.05, and an expected effect size of 0.1. The power and vice versa, may be important in chronically ill patients, and analysis gave an adequate sample size of n = 10. it has been suggested that iMg may serve as an iCa antagonist Blood was aliquoted in 1 mL evacuated tubes containing and prevent iCa entry into cells during sepsis or endotoxemia. 2 lithium-­h eparin (Aquisel blood tubes, Barcelona, Spain) for an- Calcium homeostasis is further influenced by plasma magnesium alyte measurements in whole blood. For analyte measurements since secretion of the parathyroid hormone is inhibited by high in plasma, 2 mL lithium-­h eparin tubes were used (Aquisel blood concentrations of magnesium. 8,9 tubes) and after collection, blood samples were centrifugated at Monitoring and restoration of calcium and magnesium homeo- 1500 g for 5 min at room temperature, and plasma was transferred stasis is an important aspect of managing severely ill horses. Thus, to Eppendorf tubes. To obtain serum, blood was evacuated in 5 mL measuring the free forms of calcium and magnesium is important in tubes without additives (Becton Dickinson, Madrid, Spain) and clinics. The clinical meaning of iCa is widely acknowledged in human was left to coagulate for 30 minutes. Once the clot was formed, medicine, whereas the role of iMg is recognized especially in criti- samples were centrifuged at 1500 g for 10 min at room tempera- 10,11 cally ill patients. In veterinary medicine, these studies are less ture, and serum was collected in Eppendorf tubes. All samples common and mostly analyze total calcium and magnesium, although were stored in closed tubes at 4°C until use, minimizing oxygen some reports claim the usefulness of measuring iMg.12 In addition, exposure as much as possible. Just before measurements were it is important to note that veterinary samples are not always im- made, tubes were inverted several times (blood) or were vortexed mediately processed, so the knowledge about the stability of these (plasma and serum) to obtain a homogeneous solution. Sample col- analytes in collected samples is of utmost importance. lection (time 0 of the measures) was performed at 9 am, and the The ionized forms of calcium and magnesium are usually deter- overall procedure ended at 9:30–­9:40 am. Determinations were mined using ion-­selective electrodes (ISE) analyzers that selectively performed every hour during the first 8 hours and after 24, 48, | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SANMARTÍ et al. 3 and 168 hours of storage. Additionally, three aliquots of plasma P-­values <0.05 were considered significant, presented as *P < 0.05, obtained at 0 hours were stored frozen at −20°C for ~168 h and an- **P < 0.01, and ***P < 0.001. alyzed like the refrigerated samples. Care was taken regarding the time of collection since the circadian rhythm has been acknowl- edged in humans for both calcium7 and magnesium. 3 Samples with 3 | R E S U LT S hemolysis were discarded since red blood cells contain approxi- mately three times the concentration of Mg in serum. 2 3 .1 | Effect of storage time on ionized and total Ca In the second experiment, the potential interference by hepa- and Mg in different sample types rin was evaluated by analyzing iCa and iMg in whole blood sam- ples with decreasing dilutions of sodium heparin. To simulate a Whole blood, plasma, and serum iCa and iMg concentrations did not widely used procedure in equine clinics, homemade heparin sy- change significantly over 168 hours (7 days) when kept refrigerated ringes were used to collect blood for subsequent determination at 4°C (Figure 1A,C). Similar results were obtained for tCa and tMg in of blood gases and electrolytes. The preparation consists of the plasma and serum over 7 days (Figure 1B,D). The ratios iCa/tCa and partial filling of a 1 or 2 mL syringe with commercial Na heparin iMg/tMg (Figure 1E,F) did not change either, except for serum iCa/ (5000 IU/mL) (Laboratorios Farmacéuticos Rovi, Madrid, Spain) tCa, which decreased significantly after 168 hours compared with followed by complete injection back into the vial, leaving a resid- the time of collection (t = 0; P < 0.05). In addition, the storage of ual volume within the needle hub. The residual heparin within the plasma samples for 7 days (168 hours) at −20°C did not result in signif- tip of the syringe and needle hub is considered enough to avoid icant differences in iCa (1.66 ± 0.07 and 1.61 ± 0.05 mmol/L at 0 and blood coagulation. In preliminary studies, using a precision scale 7 days) or iMg concentrations (0.52 ± 0.06 and 0.49 ± 0.06 mmol/L and weighing before and after prefilling, the volume of residual at 0 hours and 7 days, respectively; mean ± SD). Variability between fluid in a 2 mL syringe was estimated as 127 μL. The final heparin samples from different animals was higher in serum than in plasma concentration once the syringe is filled with 2 mL of blood was or blood. calculated as 297.6 IU/mL. Serial dilutions (1:10, 1:20, and 1:30) Additionally, the concentrations of iCa, iMg, tCa, and tMg in the of commercial Na heparin (5000 IU/mL) in 0.9% NaCl were used three different sample types were compared (Table 1). The concen- to similarly prefill syringes to obtain final concentrations of 29.7, trations of tCa, iMg, tMg, and the ratios iCa/tCa and iMg/tMg were 14.9, and 9.9 IU/mL, respectively, once filled with 2 mL of blood. In similar in all samples, showing low variability after sample manipu- addition, for comparison purposes, blood samples were collected lation. The concentration of iCa was lower in plasma than in serum in parallel with Li-­h eparin vacutainer tubes. (P < 0.01). Sample analysis was carried out using the Stat Profile Prime Although precautions were taken to keep the samples in anaer- Plus Vet Analyzer (Nova Biomedical), following the instructions obic conditions, pH was higher in serum and plasma than in blood of the manufacturer, which is based on ISE technology, as stated (Table 1). An increase in pH after 8 hours was observed in serum above. tCa and tMg were measured by colorimetric methods in the and plasma (P < 0.05), and a decrease in whole blood at 168 hours Beckman Coulter AU400 analyzer (Hamburg, Germany) with the (P < 0.05) (Figure 2). Figure 2 shows no apparent relationship be- Arsenazo III method (OSR 60117) and the xylidyl blue method (OSR tween pH and iCa or iMg throughout the study since both ions re- 6189), respectively. mained stable in serum and plasma. Nevertheless, the correlation Statistical analysis was performed using GraphPad Prism 7 soft- analysis showed a negative correlation between iCa vs pH in plasma ware. For all determinations, Rout tests using Q = 1% were per- (Figure 3A, R = −0.3554, P < 0.001), whereas it did not reach signif- formed to identify possible outliers. Data distribution was evaluated icance in serum (Figure 3B). There was no correlation between iMg using Shapiro–­Wilk tests. To evaluate differences over time, com- vs pH in serum or plasma in this pH range (Figure 3C,D). However, paring each type of sample, iCa, iMg, tMg, tCa, pH, Na, K, and Cl a clear negative correlation with pH was observed with the ratios were analyzed using Two-­Way ANOVA tests. Factors “time,” “type iCa/tCa and iMg/tMg in both plasma and serum (r = −[0.363–­0.674], of sample,” and their interaction “time-­t ype of sample” were evalu- P < 0.001) (Figure 3E–­H). ated. Multiple comparisons were carried out using Dunnett tests to identify simple and main effects. Heparin interference experiments were analyzed using Brown–­Forsythe and Welch ANOVA tests and 3 . 2 | Heparin interference on the determination of Dunnett T3 tests for multiple comparisons. The same analysis was iCa and iMg carried out to evaluate possible differences over time in blood anal- ysis measurements for tBil, Hct, and O2HB. Kruskal–­Wallis tests, Heparin interference was evaluated in blood and plasma, compar- using Dunn's tests for multiple comparisons, were performed to ing homemade Na-­heparin syringes at different dilutions versus evaluate pCO2, pO2, SO2, tHb, and COHb variations. Pearson cor- Li-­heparin vacutainer tubes. In blood, interference by heparin was relations were performed to evaluate possible associations between detected when comparing commercial Li-­heparin tubes (20–­30 IU pH and iCa, tCa, iMg, tMg, and iCa/tCa, or iMg/tMg using a 95% per- Li heparin/mL) with homemade syringes with high concentration cent of confidence. Descriptive data are presented as the mean ± SD. (~200–­300 IU Na heparin/mL), whereas no effect was observed | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 SANMARTÍ et al. F I G U R E 1 Effect of storage at 4°C up to 168 hours on measured concentrations of (A) ionized Ca (iCa), (B) total Ca (tCa), (C) ionized Mg (iMg), (D) total Mg (tMg), (E) ratio iCa/tCa, and (F) ratio iMg/tMg of samples obtained from 10 horses in serum (blue), heparin-­plasma (green), and whole blood (red) hours and using the Prime Vet Plus (Nova Biomedical) analyzer (iCa and iMg) and the AU400 analyzer (tCa and tMg). Data are presented as mean and standard deviation. For each type of sample, Rout tests with Q = 1% were carried out to identify outliers. Data distribution was evaluated using Shapiro–­Wilk tests. Two-­way ANOVA tests were performed to compare variations within each sample and between sample types. Factor “time” and “type of sample” and their interaction were evaluated. For multiple comparisons, Dunnett tests were performed using a sample at time 1 as the control. Serum iCa/tCa was significantly different at 168 hours of storing versus time 1 (*P < 0.05). | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SANMARTÍ et al. 5 TA B L E 1 Concentrations of ionized and total calcium and magnesium and their ratios in whole blood, plasma, and serum samples. Minimum Maximum Number of Analyte Type of sample Mean ± SD (mmol/L) (mmol/L) (mmol/L) samples (n) iCa Blood (Prime+Vet) 1.61 ± 0.08a,b 1.40 1.76 110 Plasma (Prime+Vet) 1.58 ± 0.10 b 1.34 1.74 110 a Serum (Prime+Vet) 1.64 ± 0.20 1.35 2.18 109 tCa Plasma (Olympus) 2.85 ± 0.17 2.35 3.11 95 Serum (Olympus) 2.89 ± 0.39 2.48 4.32 95 iMg Blood (Prime+Vet) 0.51 ± 0.05 0.41 0.61 110 Plasma (Prime+Vet) 0.50 ± 0.06 0.38 0.60 110 Serum (Prime+Vet) 0.51 ± 0.05 0.41 0.60 110 tMg Plasma (Olympus) 0.67 ± 0.12 0.49 1.26 95 Serum (Olympus) 0.64 ± 0.10 0.45 0.93 95 iCa/tCa Plasma (Prime+Vet) 0.55 ± 0.02 0.51 0.60 95 Serum (Olympus) 0.56 ± 0.03 0.50 0.63 95 iMg/tMg Plasma (Prime+Vet) 0.78 ± 0.07 0.47 0.93 95 Serum (Olympus) 0.81 ± 0.11 0.54 0.98 95 pH Blood (Prime+Vet) 7.39 ± 0.05d 7.18 7.51 106 Plasma (Prime+Vet) 7.66 ± 010 c 7.50 7.99 104 Serum (Prime+Vet) 7.67 ± 0.12c 7.38 7.98 92 Note: Data are presented in mmol/L by mean ± standard deviation (SD). Minimum and maximum values, the number of samples and P-­values comparing each sample type for each analyte were also defined. Superindexes: a ≠ b: P < 0.01; c ≠ d: P < 0.01. Abbreviations: iCa, ionized Ca; iMg, ionized Mg; tCa, total Ca; tMg, total Mg. with syringes with low heparin concentrations (9.9 up to 30 IU Na was significant at 168 h (P < 0.05). Serum and plasma glucose and heparin/mL). Concentrations of iCa and iMg decreased by ~20% in lactate did not change significantly. samples using undiluted heparin compared with commercial tubes Finally, blood-­gas analysis measurement in samples collected (P < 0.01 for iCa and iMg) (Figure 4). with vacutainer tubes was performed for exploratory purposes. High stability was observed for up to 48 hours in partial pressure carbon dioxide (pCO2), partial pressure of oxygen (pO2), total hemo- 3 . 3 | Effect of storage time on other globin (tHb), oxyhemoglobin (O2Hb), carboxyhemoglobin (COHb), electrolytes and biochemical parameters oxygen saturation (sO2), total bilirubin (tBil), and hematocrit (Hct) (Figure S3). The Stat Profile Prime Plus Vet equipment also allows the quantifica- tion of other parameters in blood, plasma, or serum samples, whose stability over time was also examined. 4 | DISCUSSION Monovalent ions (Na, Cl, and K) showed good stability up to 48 hours with low variability, except for K in whole blood, as ex- Determining the ionized forms iCa and iMg, in addition to tCa and pected (Figure S1A–­C). The concentrations of Na and Cl were sig- tMg, is increasingly important since these analytes are physiologi- nificantly higher in serum than in plasma or blood (136.10 ± 1.96, cally important and do not always correlate with the total concentra- 132.50 ± 2.78, and 133.00 ± 2.05 mmol/L for Na and 106.60 ± 2.04, tion. Both iMg and iCa are likely physiologically interdependent, and 104.30 ± 2.20, and 102.90 ± 1.92 mmol/L for Cl, respectively, being in equine patients, both hypocalcemia and hypomagnesemia have P < 0.001 for all comparisons described). The concentration of K been associated with decreased survival in critical illness.5,6,13,14 was stable in serum and plasma over time (4.02 ± 0.39 for serum and In veterinary medicine, the determination of iCa and iMg in whole 3.82 ± 0.12 mmol/L for plasma) and progressively increased in blood blood is not always possible and, in addition, veterinary samples samples from 3.82 ± 0.19, 4.23 ± 0.33, 4.87 ± 0.58, 5.39 ± 0.79, and are not always immediately processed, so the knowledge about 7.81 ± 1.50 mmol/L by 0, 8, 24, 48 and 168 hours, respectively (time the stability of these analytes and a comparison of these analytes 0 vs 24, 48, and 168 hours; P < 0.001). between different sample types are important issues. The results Additionally, the stability of glucose and lactate in the three sam- of the present study have shown that iCa, iMg, and the total forms ple types was evaluated (Figure S2A,B, respectively). In whole blood, (tCa, tMg) and the ratios iCa/tCa and iMg/tMg are stable up to a decrease in glucose and an increase in lactate was observed, which 168 hours. Furthermore, concentrations are similar among sample | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 SANMARTÍ et al. types, except for iCa, which was slightly lower in plasma. Freezing and iCa/tCa = 0.52 ± 0.02% that were similar to our results. In at −20°C does not affect these parameters. Electrolytes (Na, K), glu- the current work, similar concentrations were found for the three cose, lactate, and blood gases are stable in plasma and serum. These sample types (blood, plasma, and serum), except iCa concentration results facilitate the use of these analytes in the clinical diagnosis of in plasma, which was slightly but significantly lower, similar to the equine diseases. On the other hand, caution must be exercised when previous authors. Other authors have found lower iMg concen- using homemade heparin syringes, due to the potential interference trations in heparinized plasma compared with serum.16 The ratio of heparin. of the ionized vs total form was 0.55 for calcium, similar to values Compared with the literature, values for iMg and iCa con- previously described,1,2,15 and 0.80 for magnesium, slightly higher 6,15,16 centrations are similar to those described in adult horses. than with other studies.15 Berlin et al.15 determined the concentrations of iCa and iMg in The use of serum and plasma instead of whole blood and the whole blood, serum, and plasma, and tCa and tMg in the serum effect of the storage time on iCa and iMg are relevant questions, of healthy horses. These authors reported concentrations especially when dealing with horses since a clinical pathology lab- of iMg = 0.48–­0 .52 mmol/L depending on the sample type, oratory is not always immediately available. The results presented iCa = 1.61–­1.69 mmol/L, and ratios of iMg/tMg = 0.64 ± 0.07% here indicate that stability is high for the ionized forms of both calcium and magnesium. In samples kept at 4°C for 168 hours, a slight nonsignificant decrease in iMg (2.5%, 5.4%, and 8.5%) and iCa (1.9%, 1.7%, and 4.6%) concentrations, respectively, were observed. This is similar to results described by Lopez et al. in a micropartition study, where the authors found that refrigeration (4°C) did not affect iCa values, whereas iMg declined by 8% after 120 hours.17 For the total forms, there is a nonsignificant decrease of 3.9 and 3.4% at 168 h for tCa in plasma and serum, respectively, and no change for tMg. It is well-­k nown that iCa and iMg concentrations are influenced by the acid–­b ase status of the patient. Acidosis increases iCa and iMg concentrations, whereas alkalosis reduces them.1,2 This prop- erty is clinically important when treating animals with respiratory or metabolic alkalosis, often observed after prolonged strenuous endurance exercise. iCa and iMg concentrations are also altered with changes in the pH of the collected samples, which can hap- pen during processing. In our case, although preventive measures were taken to minimize the aerobic exposure of plasma in serum samples, an increased pH in serum and plasma was observed due to the loss of CO2 over time, although the increase in pH was only significant after 8 hours. Whole blood is better able to maintain F I G U R E 2 Effect of storage at 4°C up to 168 hours on pH and the pH due to its physiologic buffering systems. Our results show its relation with ionized calcum (iCa) and ionized magnesium (iMg) that, despite the steadily increased pH in serum and plasma, the in whole blood (red), plasma (blue), and serum (green). Samples of influence on concentrations of the ionized forms of calcium and 10 horses were measured with Prime Vet Plus (Nova Biomedical) magnesium is not very relevant in this pH range (pH = 7.6–­7.8). analyzer. Data are presented as mean and standard deviation. For each type of sample, Rout tests with Q = 1% were carried out to However, a close look at the correlation between iCa and pH re- identify outliers. Data distribution was evaluated using Shapiro–­ vealed a significant negative correlation in plasma that was mild Wilk tests. Two-­way ANOVA tests were performed to compare and nonsignificant in serum. On the other hand, there was no ap- variations within each sample and between sample types. Factor parent correlation between iMg and pH. The negative correlation “time” and “type of sample” and their interaction were evaluated. is explained because iCa and iMg bind to anionic protein binding For multiple comparisons, Dunnett tests were performed using a sample at time 1 as the control. Serum pH increased significantly sites and this binding increases at high pH, causing lower values after 8 hours of sample collection (P < 0.05, blue asterisks). Blood for both ions. When looking at the iCa/tCa and iMg/tMg ratios, pH decreased significantly after 168 hours (P < 0.01, red asterisk). the negative correlation with pH is stronger, further supporting F I G U R E 3 Pearson correlations of ionized Ccalcum (iCa) and ionized magmnesium (iMg) concentrations, and the iCa/tCa and iMg/tMg ratios versus pH in plasma and serum. Correlations were calculated using a 95% confidence interval. Pearson coefficient (r) and P-­values are described in each graph. (A) Correlation of plasma pH versus iCa; (B) Correlation of serum pH versus iCa; (C) Correlation of plasma pH versus iMg; (D) Correlation of serum pH versus iMg; (E) Correlation of plasma pH versus iCa/tCa ratio; (F) Correlation of serum pH versus iCa/tCa ratio; (G) Correlation of plasma pH versus iMg/tMg ratio; (H) Correlation of serum pH versus iMg/tMg ratio. 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License |7 SANMARTÍ et al. | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13200 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [11/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 SANMARTÍ et al. Finally, regarding the other biochemical parameters, the con- centrations of electrolytes (Na, K, Cl) were similar to that described previously. 21,22 Our results confirm that the three electrolytes were stable in plasma and serum when cells have been removed from the sample, and there was no hemolysis. However, the levels of K in whole blood markedly increased due to hemolysis after collection since K is mostly an intracellular cation. Glucose and lactate were also stable in plasma and serum, and as expected, glucose decreased in whole blood throughout the time since it was converted to lactate mainly by the glycolytic pathway in erythrocytes (which lack mitochondria and are very dependent on glucose as a nutrient). As a consequence, lactate increased in whole blood with time. Regarding blood gases, pO2, pCO2 and total and oxygenated he- moglobin had good stability throughout the time. It has to be re- marked that samples were kept in the cold and tightly closed and consequently in anaerobic conditions. 5 | CO N C LU S I O N S Samples stored at 4°C can be used to determine iCa, iMg, and other parameters up to 8 hours after collection. Heparin interference F I G U R E 4 Effect of heparin concentration in plasma on gives lower values for iCa and iMg and should be considered if using measured concentrations of (A) ionized Ca (iCa), (B) total Ca (tCa), homemade heparin syringes instead of commercial heparinized sy- (C) ionized Mg (iMg), and (D) total Mg (tMg) in equine heparinized plasma samples obtained from 5 different horses, using the ringes or evacuated tubes. Prime Plus Vet (Nova Biomedical) analyzer (iCa and iMg) and the AU400 analyzer (tCa and tMg). Blood samples were collected AC K N OW L E D G M E N T S in commercial Li-­heparin tubes (Hep-­Li; heparin concentration The authors thank Nova Biomedical Corporation for providing the 20 UI/mL), and heparin-­flushed syringes at different Na-­heparin Stat Profile Prime Plus Vet analyzer and associated reagents used concentrations (blue, Na-­heparin syringe nondiluted 297 IU/mL; for this study. red, Na heparin 29.7 IU/mL; green, Na heparin 14.9 IU/mL; purple, Na heparin 9.9 IU/mL). Samples were analyzed immediately after collection. Rout tests with Q = 1% were carried out to identify D I S C LO S U R E outliers. Data distribution was analyzed using Shapiro–­Wilk The authors have indicated that they have no affiliations or final in- tests. Brown–­Forsythe and Welch ANOVA tests and Dunnett T3 volvement with any organization or entity with a financial interest tests were performed to evaluate possible differences between in, or in financial competition with the subject matter discussed in groups and to make multiple comparisons, respectively. *P < 0.05; **P < 0.01. this article. ORCID this mechanism. These results reinforce the data reported in Anna Bassols https://orcid.org/0000-0003-4213-2274 horses on the influence of pH, which is quite robust for iCa1 but scarcer for iMg. 2 REFERENCES Our results show that heparin interferes with the determination 1. Toribio RE. Disorders of calcium and phosphate metabolism of iCa and iMg at high concentrations (~200–­300 IU/mL) causing de- in horses. Vet Clin North Am -­Equine Pract. 2011;27:129-­147. doi:10.1016/j.cveq.2010.12.010 creased values of both parameters of around 20%. Although this has 2. Stewart AJ. Magnesium disorders in horses. 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